1. Field of the Invention
The present invention relates to novel phenylalanine derivatives that are inhibitors of α4 (including α4β7 and α4β1) mediated adhesion which could be useful in treating conditions such as asthma, diabetes, rheumatoid arthritis, inflammatory bowel disease and other diseases involving leukocyte infiltration to the gastrointestinal tract or other epithelial lined tissues; such as, skin, urinary tract, respiratory airway and joint synovium.
The inhibitors of the present invention could also be useful in treating conditions involving leukocyte infiltration to other tissues including lung, blood vessels, heart and nervous system as well as transplanted organs such as kidney, liver, pancreas, heart and intestine, and blood vessels.
2. Description of the Related Art
The adhesion of leukocyte to endothelial cells or extracellular matrix proteins is a fundamental process for immunity and inflammation and involves multiple adhesive interactions. The earliest events in this process include leukocyte rolling followed by changes in integrin avidity, which lead to subsequent firm adhesion (for reviews see Butcher, Cell 67:1033-1036 (1991); Harlan, Blood 3:513-525 (1985); Hemler, Annu. Rev. Immunol. 8:365-400 (1990); Osborn, Cell 62:3-6 (1990); Shimizu et al., Immunol. Rev. 114:109-143 (1990); Springer, Nature 346:425-434 (1990); and Springer, Cell 76:301-314 (1994)). In response to chemotactic factors, the leukocytes must migrate through two adjacent endothelial cells and into tissues that are composed, in part, of the extracellular matrix protein fibronectin (FN) (see Wayner et al., J. Cell Biol. 105:1873-1884 (1987)) and collagen (CN) (see Bornstein et al., Ann. Rev. Biochem. 49:957-1003 (1980); and Miller, Chemistry of the collagens and their distribution, in “Extracellular Matrix Biochemistry”, K. A. Piez and A. H. Reddi, editors, Elsevier, Amsterdam, 41-78 (1983)). Important recognition molecules that participate in these reactions belong to the integrin gene superfamily (for reviews see Hemler, Annu. Rev. Immunol. 8:365-400 (1990); Hynes, Cell 48:549-554 (1987); Shimizu et al., Immunol. Rev. 114:109-143 (1990); and Springer, Nature 346:425-434 (1990)).
Integrins are heterodimers composed of non-covalently associated subunits, referred to as the alpha (α) and beta (β) subunits (for reviews see Hemler, Annu. Rev. Immunol. 8:365-400 (1990); Hynes, Cell 48:549-554 (1987); Shimizu et al., Immunol. Rev. 114:109-143 (1990); and Springer, Nature 346:425-434 (1990)). To date, 8 integrin β subunits have been identified which can associate with 16 distinct a subunits to form 23 distinct integrins. The α4β1 integrin, also known as VLA-4 (Very Late Antigen-4), is expressed on a variety of cells including lymphocytes, monocytes and eosinophils (see Hemler et al., J. Bio. Chem. 262:11478-11485 (1987); and Bochner et al., J. Exp. Med. 173:1553-1556 (1991)) and may have an important role in the recruitment of these cells during inflammation. VLA-4 is a receptor for vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., Cell 60:577-584 (1990)) and the connecting segment 1 (CS-1), an alternatively spliced region of the FN A chain (Wayne et al., J. Cell Biol. 109:1321-1330 (1989)). The β7 integrin subunit, first cloned by Erle et al. (Erle et al., J. Biol. Chem. 266:11009-11016 (1991)), is expressed only on leukocytes and is known to associate with two distinct a subunits, α4 (Ruegg et al., J. Cell Biol. 117:179-189 (1992)) and αE (Cerf-Bensussan et al., Eur. J. Immunol. 22:273-277 (1992); and Kilshaw et al., Eur. J. Immunol. 21:2591-2597 (1991)).
The α4β7 complex has three known ligands (VCAM-1, CS-1, MAdCAM-1). One ligand which shows unique specificity for α4β7 is Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) (see Andrew et al., J. Immunol. 153:3847-3861 (1994); Briskin et al., Nature 363:461-464 (1993); and Shyjan et al., J. Immunol. 156:2851-2857 (1996)). MAdCAM-1 is highly expressed on Peyer's patch high endothelial venules, in mesenteric lymph nodes, and on gut lamina propria and mammary gland venules (Berg et al., Immunol. Rev. 105:5-18 (1989)). Integrin α4β7 and MAdCAM-1 have been shown to be important in regulating lymphocyte trafficking to normal intestine (Holzmann et. al., Cell 56:37-46 (1989)).
The second ligand for α4β7 is CS-1 (see Guan et al., Cell 60:53-61 (1990); and Wayner et al., J. Cell Biol. 109:1321-1330 (1989)). The cell-binding site within CS-1 is composed of 25 amino acids where the carboxy terminal amino acid residues, BILDVPST, form the recognition motif (see Komoriya et al., J. Biol. Chem. 266:15075-15079 (1991); and Wayner et al., J. Cell Biol. 116:489-497 (1992)).
The third ligand for (α4β7 is vascular cell adhesion molecule-1 (VCAM-1), a cytokine inducible protein expressed on endothelial cells (see Elices et al., Cell 60:577-584 (1990); and Ruegg et al., J. Cell Biol. 117:179-189 (1992)). It remains to be unequivocally shown whether MAdCAM-1, VCAM-1 and CS-1 bind to the same site on α4β7. Using a panel of Monoclonal antibodies, Andrew et al. showed that α4β7 interaction with its three ligands involves distinct but overlapping epitopes (Andrew et al., J. Immunol. 153:3847-3861 (1994)). VCAM-1 and CS-1 (see Elices et al., Cell 60:577-584 (1990)) are two ligands which are shared by α4β7 and α4β1. In addition, α4β1 is also known to bind to osteopontin, a protein upregulated in arteriosclerotic plaques (see Bayless et al., J. Cell Science 111:1165-1174 (1998)).
Utility of the Invention
A number of in vivo studies indicate that the α4 integrins (α4β1/(α4β7) play a critical role in the pathogenesis of a variety of diseases. Monoclonal antibodies directed against α4 have been tested in a variety of disease models. Efficacy of anti-α4 antibody was demonstrated in rat and mouse models of experimental autoimmune encephalomyelitis (see Baron et al., J. Exp. Med. 177:57-68 (1993); and Yednock et al., Nature 356:63-66 (1992)). A significant number of studies have been done to evaluate the role of α4 in allergic airways (see Abraham et al., J. Clin. Invest. 93:776-787 (1994); Bochner et al., J. Exp. Med. 173:1553-1556 (1991); Walsh et al., J. Immunol. 146:3419-3423 (1991); and Weg et al., J. Exp. Med. 177:561-566 (1993)). For example, monoclonal antibodies to α4 were effective in several lung antigen challenge models (see Abraham et al., J. Clin. Invest. 93:776-787 (1994); and Weg et al., J. Exp. Med. 177:561-566 (1993)). The cotton-top tamarin, which experiences spontaneous chronic colitis, showed a significant attenuation of colitis when anti-α4 antibody or anti-α4β7 antibody was administered (see Bell et al., J. Immunol. 151:4790-4802 (1993); Podolsky et al., J. Clin. Invest. 92:372-380 (1993); and Hesterberg et al., Gastroenterology 111:1373-1380 (1996)). In scid mice reconstituted with CD45RBhigh CD4+ T cells, monoclonal antibodies to β7 or MAdCAM-1 blocked recruitment of lymphocytes to the colon and reduced the severity of inflammation in the colon as judged histologically (see Picarella et al., J. Immunol. 158:2099-2106 (1997)). Monoclonal antibodies to α4 inhibit insulitis and delay the onset of diabetes in the non-obese diabetic (NOD) mouse (see Baron et al., J. Clin. Invest. 93:1700-1708 (1994); Burkly et al., Diabetes 43:529-534 (1994); and Yang et al., Proc. Natl. Acad. Sci. USA 90:10494-10498 (1993)). Other diseases where α4 has been implicated include rheumatoid arthritis (see Laffon et al., J. Clin. Invest. 88:546-552 (1991); and Morales-Ducret et al., J. Immunol. 149:1424-1431 (1992)), atherosclerosis (see Cybulsky et al., Science 251:788-791 (1991)), allograft rejection (Isobe et al., J. Immunol. 153:5810-5818 (1994)), and nephritis (Allen et al., J. Immunol. 162:5519-5527 (1999)). Delayed type hypersensitivity reaction (see Issekutz, J. Immunol. 147:4178-4184 (1991)), contact hypersensitivity response (see Chisholm et al., Eur. J. Immunol. 23:682-688 (1993); and Ferguson et al., J. Immunol. 150:1172-1182 (1993)) and intimal hyperplasia (Lumsden et al., J. Vasc. Surg. 26:87-93 (1997)) are also blocked by anti-α4 antibodies. For an excellent review of in vivo studies implicating α4 in disease, see Lobb et al., J. Clin. Invest. 94:1722-1728 (1995).
Leukocyte adhesion to inflamed synovium was suggested to be dominated by α4β1/VCAM-1 interactions, however, increased numbers of α4β7 positive T cells were also found in the synovial membrane of rheumatoid arthritis patients (McMurray, Semin. Arthritis Rheum. 25:215-233 (1996)) and it was suggested that the augmented expression of α4β7 may contribute to the development and perpetuation of this disease (see Lazarovits et al., J. Immunol. 151:6482-6489 (1993)). In the NOD mouse, MAdCAM-1 was expressed on high endothelial venules in inflamed islets within the pancreas suggesting a role for α4β7 in diabetes (see Yang et al., Diabetes 46:1542-1547 (1997)). The expression of α4β1/β4β7 on a variety of leukocytes and the presence of α4β1/α4β7 positive cells in diseased tissues imply that the two receptors may play important roles in cellular recruitment to a number of sites of inflammation. For example, monoclonal antibodies to α4 were effective in several lung antigen challenge models such as ovalbumin-induced asthma in mice, rats and guinea-pigs (See Pretolani et al., J. Eyp. Med. 180: 795-805 (1994), Fryer et al., J. Clin. Invest. 99:2036-2044 (1997); and Henderson et al., J. Clin. Invest. 100: 3083-3092 (1997)). The expression of α4β7 and α4β1 on lymphocytes and eosinophils, together with in vitro studies showing that (α4β7/α4β1 mediates human eosinophil adhesion to VCAM-1, CS-1 and MAdCAM-1 (Walsh et al., Immunology 9:112-119 (1996)), suggests that α4 is a suitable therapeutic target for the treatment of asthma. Collectively, these data suggest that integrins α4β7 and α4β1 may play an important role in a variety of inflammatory diseases.
The use of monoclonal antibodies against integrins in vivo has demonstrated that a number of integrins are indeed valid therapeutic targets for inflammatory, immune-mediated diseases, cardiovascular diseases and in organ transplantation.
Also, it has been described that an orally bioavailable, non-peptide small molecule antagonist of α4 could be useful in treating or preventing conditions such as asthma, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis and other diseases (see WO99/36393).
The objective here was to define an orally bioavailable and potent small molecule antagonist of α4 integrins. Small molecules that are potent inhibitors of α4 mediated adhesion to either MAdCAM-1, VCAM-1, or CS-1 and which could be useful for the treatment or prevention of inflammatory diseases and/or allergic diseases are disclosed.